A major challenge facing the seafood industry is confirming the identity of fish sold in restaurants, seafood markets, and by wholesalers. Mislabeling of seafood products, such as substituting more valuable species with less valuable ones, is a growing problem. (Trotta, et al.; Multiplex PCR methods for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. J Agric Food Chem. 2005; 53: 2039-2045; Espiñeira, et al., Authentication of anglerfish species (Lophius spp) by means of polyermase chain reaction-restriction fragment length polymorphism (PCR-RFLP) and forensically informative nucleotide sequencing (FINS) methodologies. J Agric Food Chem. 2008; 56: 10594-10599; Wen, et al., The application of PCR-RFLP and FINS for species identification used in sea cucumbers (Aspidochirotide: Stichopodidae) products from the market. Food Control. 2010 April; 21(4):403-407; Ortea, et al., Closely related shrimp species identification by MALDI-ToF mass spectrometry. J Aquatic Food Prod Technol. 2009; 18: 146-155; Espiñeira, et al., Development of a method for the identification of scombroid and common substitute species in seafood products by FINS. Food Chem. 2009; 117: 698-704). A recent study found that as much as 77% of red snapper samples were mislabeled (Trotta, et al.; Multiplex PCR methods for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. J Agric Food Chem. 2005; 53: 2039-2045).
Grouper species from the Epinephelus and Mycteroperca genera are in heavy demand (Trotta, et al.; Multiplex PCR methods for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. J Agric Food Chem. 2005; 53: 2039-2045). Due to such high demand, sporadic availability, and therefore cost, grouper is commonly substituted with Nile perch (Lates niloticus) and wreck fish (Polyprio americanus) (Trotta, et al.; Multiplex PCR methods for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. J Agric Food Chem. 2005; 53: 2039-2045). In Florida, the Department of Agriculture and Consumer Services has uncovered several instances of grouper substitution, including the discovery of almost 8,000 pounds of Vietnamese broadhead fillets marked for sale as grouper (McElroy, Department Press Release Bronson Announces Discovery of Nearly 8,000 Pounds Of Bogus Grouper. FL Department of Agriculture and Consumer Services. Released May 9, 2006). An investigation into grouper menu items in Tampa Bay area restaurants revealed substituted species to be emperor (generally a Lethrinus spp.), hake (typically Urophycis or Merluccius spp.), sutchi (Pangasius hypophthalmus), bream (numerous species are considered bream), green weakfish (Cynoscion virescens) and painted sweetlips (Diagramma pictum) (Copes, Attorney General Bill McCollum News Release: Attorney General, Tampa Restaurants Reach Agreement Over Fish Substitutions. Office of the Attorney General of Florida. Released Mar. 9, 2007), tilapia, bream, ponga, hake, and emperor fish as grouper “impostors” (Nohlgren, State finds more grouper impostors. St. Petersburg Times, St. Petersburg, Fla. Jan. 30, 2007, pg. A1).
Identification of seafood in the marketplace after the distinguishing external characteristics are removed (skin, fins, head, etc) is problematic; the flesh of many fish is similar enough that it can be difficult to identify the source (Wen, et al., The application of PCR-RFLP and FINS for species identification used in sea cucumbers (Aspidochirotide: Stichopodidae) products from the market. Food Control. 2010;:403-407; Espiñeira, et al., Development of a method for the identification of scombroid and common substitute species in seafood products by FINS. Food Chem. 2009; 117: 698-704; Aguilera-Muñoz, et al., Authentication of commercial chilean mollusks using ribosomal internal transcribed spacer (ITS) as specie-specific DNA marker. Gayana. 2008; 72(2): 178-187; Yancy, et al., Potential use of DNA barcodes in regulatory science: applications of the regulatory fish encyclopedia. J Food Protection. 2008; 71(1): 210-217).
In Florida, the Department of Agriculture and Consumer Services has uncovered several instances of grouper substitution, including the discovery of almost 8,000 pounds of Vietnamese broadhead fillets marked for sale as grouper (McElroy, Department Press Release: Bronson Announces Discovery of Nearly 8,000 Pounds Of Bogus Grouper. FL Department of Agriculture and Consumer Services Released. May 9, 2006). The Florida Attorney General's office has settled several complaints against restaurants and wholesalers. An investigation into grouper menu items in Tampa Bay area restaurants revealed substituted species to be emperor (generally a Lethrinus spp.), hake (typically Urophycis or Merluccius spp.), sutchi (Pangasius hypophthalmus), bream (numerous species are considered bream), green weakfish (Cynoscion virescens) and painted sweetlips (Diagramma pictum) (Copes, Attorney General Bill McCollum News Release: Attorney General, Tampa Restaurants Reach Agreement Over Fish Substitutions. Office of the Attorney General of Florida. Released Mar. 9, 2007). Additional media investigations have revealed tilapia, bream, ponga, hake, and emperor fish as grouper “impostors” (Nohlgren, State finds more grouper impostors. St. Petersburg Times, St. Petersburg, Fla. Jan. 30, 2007, pg. A1). Fines or settlements imposed to local restaurants and wholesale suppliers for intentionally or unintentionally serving or selling other fish for grouper are costly (Nohlgren, Payment ends inquiry into bogus grouper. St. Petersburg Times, St. Petersburg, Fla. Sep. 4, 2008, pg. A1); a recent settlement between the Florida Attorney General and a wholesaler in Florida was $300,000 (Copes, McCollum: Settlement Reached with National Food Distributor over Grouper Allegations. Office of the Attorney General of Florida. Released Sep. 3, 2008). Variable stocks and state and federal grouper catch regulations that are becoming more restrictive, and can be confusing (Tomalin, Changes bring confusion: As federal and state agencies tweak grouper regulations, recreational anglers scratch heads over rules and reasons. St. Petersburg Times, St. Petersburg, Fla. Jan. 9, 2009, pg. C8), may lead to more attempts at substitution of other fish products for grouper. Identification of the fish in the marketplace after the distinguishing external characteristics are removed (skin, fins, head, etc) is problematic; the flesh of many fish is similar enough that it can be difficult to identify the source. Various protein-based techniques including electrophoretic, immunologic, and chromatographic methods have been employed to identify fish fillets. The immunologic methods are best suited for analyzing large numbers of samples, whereas electrophoretic and chromatographic methods are complex and require facilities and instrumentation often not routinely available to seafood regulatory agencies (Rasmussen and Morrissey, DNA-Based methods for the identification of commercial fish and seafood species. Comprehensive Reviews in Food Science and Food Safety. 2008; 7: 280-295). While protein isoelectric focusing has been the standard method in use by the U.S. Food and Drug Administration (FDA) in the past, current methods are focused on nucleic acid-based technology.
Biochemical assays are being developed to aid in the identification of various seafood species, with many relying on the 16S ribosomal RNA gene or cytochrome oxidase I (COI) gene (Wen, et al., The application of PCR-RFLP and FINS for species identification used in sea cucumbers (Aspidochirotide: Stichopodidae) products from the market. Food Control. 2010 April; 21(4):403-407; Ortea, et al., Closely related shrimp species identification by MALDI-ToF mass spectrometry. J Aquatic Food Prod Technol. 2009; 18: 146-155). Further, assays are moving toward DNA-based analyses over protein-based assays, due to less degeneration of the genetic code and the presence of non-coding regions (Espiñeira, et al., Development of a method for the identification of scombroid and common substitute species in seafood products by FINS. Food Chem. 2009; 117: 698-704), though protein-based assays still find acceptance (Ortea, et al., Closely related shrimp species identification by MALDI-ToF mass spectrometry. J Aquatic Food Prod Technol. 2009; 18: 146-155). DNA-based methods afford greater resolution and speed in identifying fish species than protein-based techniques (Rasmussen and Morrissey DNA-Based methods for the identification of commercial fish and seafood species. Comprehensive Reviews in Food Science and Food Safety. 2008; 7: 280-295). Methods incorporating the polymerase chain reaction (PCR) include random amplified polymorphic DNA (DNA-RAPD; Bardakci and Skibinski, Application of the Rapd Technique in Tilapia Fish—Species and Subspecies Identification. Heredity. 1994; 73: 117-123), PCR restriction length polymorphisms (PCR-RFLP; Ram, et al. Authentication of canned tuna and bonito by sequence and restriction site analysis of polymerase chain reaction products of mitochondrial DNA. Journal of Agricultural and Food Chemistry. 1996; 44: 2460-2467), and PCR-single strand conformational polymorphism (PCR-SCCP; Asensio et al., PCR-SSCP: A simple method for the authentication of grouper (Epinephelus guaza), wreck fish (Polyprion americanus), and Nile perch (Lates niloticus) fillets. Journal of Agricultural and Food Chemistry. 2001; 49: 1720-1723). In all these methodologies, DNA is extracted from the tissue sample and purified. The PCR reaction is performed in a thermal cycler, and the resulting amplified DNA is then processed in a variety of ways, such as agarose gel to confirm presence and size, digestion to form a pattern of fragments that can be analyzed (PCR-RFLP) and compared to databases, or DNA sequencing. Current methods of identifying grouper from non-grouper fish use nucleic acid assays, such as those described by Kondo, et al. (U.S. Pat. No. 5,853,981) and Nazarenko, et al. (U.S. Pat. No. 5,866,336).
The FDA is in partnership with the Consortium for the Barcode of Life (CBOL) on research and validation of DNA bar-coding methods. This involves PCR amplification of a specific genetic locus, typically the mitochondrial cytochrome c oxidase I gene (COI), sequencing, and comparison of sequence to a database of sequences from verified, or vouchered, specimens (Yancy, et al., Potential use of DNA barcodes in regulatory science: Applications of the Regulatory Fish Encyclopedia. Journal of Food Protection. 2008; 71: 210-217). A related group, the Fish Barcode of Life Initiative (FISH-BOL), is building the bar-code database of fish species. The intended use of this methodology is to amplify a standardized fragment of the COI gene producing a 648 nucleotide product that is then sequenced and the sequence submitted to an online search engine (BOLD Systems, Barcode of Life identification engine; Biodiversity Institute of Ontario, University of Guelph, Guelph, ON) which performs the comparison to known sequences and produces an identification or close approximation. DNA bar-coding methodology has advantages, such as the use of a standardized genetic locus for characterization and standardized laboratory procedures for its amplification. The method provides a confirmed identity or at least a good approximation depending on database coverage of the species.
DNA-based methods afford greater resolution in identifying fish but are generally not workable outside the biotechnology laboratory. Further, the DNA require PCR amplification followed by some type of electrophoresis step or sequencing to confirm species identity. The size and power requirements to amplify DNA are largely dictated by the need for rapid and accurate temperature cycling. Although research is ongoing, development of a field-portable and especially hand-held PCR thermal cycler is a very technically daunting challenge.
Beyond the amplification of target sequences, DNA sequencing is even more daunting in hardware, reagent, and expertise requirements. While almost all molecular biology labs have the capability and expertise to perform PCR, sequencing is typically outsourced to relatively fewer labs with this capability, increasing the cost and turn-around time for results. A portable DNA sequencing device would be an even greater technological challenge than a portable PCR thermal cycler. Multiplex PCR and Real Time PCR have also been recently reported as methods to differentiate grouper from common substitutes (Trotta, et al. Multiplex PCR method for use in real-time PCR for identification of fish fillets from grouper (Epinephelus and Mycteroperca species) and common substitute species. Journal of Agricultural and Food Chemistry. 2005; 53: 2039-2045). Real-time PCR involves a specific probe molecule that fluoresces upon amplification of the target sequence in PCR. This method requires expensive instrumentation in the form of a real-time thermal cycler, an even more cumbersome and costly device than a standard thermal cycler. For these reasons, molecular identification of seafood has remained the purview of expensive molecular biology labs and trained lab technicians. A method to allow rapid and relatively easy identification of grouper tissues (i.e. seafood fillets) that could be performed by a variety of individuals with moderate technical skill would enable much broader testing for forensic and food quality applications. Such testing could then be performed at inspection points, fish markets, and even conceivably by restaurateurs. This technology would also be immensely beneficial to biological research employing biopsy hooks for non-lethal sampling of fishable stocks.
An alternative molecular technique termed nucleic acid sequence based amplification (NASBA) is similar to PCR in terms of amplification of specific nucleic acid sequences via an enzymatic reaction (Davey and Malek EP 0329822; Davey, et al.; WO/1991/002818). NASBA differs from PCR in some key ways however. NASBA reactions are simple and quick, with the assays taking as little as about an hour including incubation (Baeummer, et al., A rapid biosensor for viable B. anthracis spores. Anal Bioanal Chem. 2004; 380: 15-23). The system is based on an isothermal amplification protocol (i.e. does not require temperature cycling) that simplifies the hardware requirements; the process does not require a thermostable DNA polymerase or a thermal cycler, and works on RNA rather than DNA. Work using NASBA as shown the system able to target and quantify a series of microbial RNAs in the marine environment (Casper et al. Detection and quantification of the red tide dinoflagellate Karenia brevis by real-time nucleic acid sequence-based amplification. Appl Environ Microbiol. 2004; 70: 4727-4732; Casper et al. Development and evaluation of a method to detect and quantify enteroviruses using NASBA and internal control RNA (ICNASBA). J. Virol. Methods. 2005; 124: 149-155; Patterson, et al., A nucleic acid sequence-based amplification assay for real-time detection of norovirus genogroup II. J. Appl. Microbiol. 2006; 101: 956-963).
Thus, while DNA barcode amplification and sequencing will likely continue to be advanced as a conclusive tissue identification technique, the complexity of this procedure precludes use outside a molecular biology laboratory for the near future. However, with a simple yet robust sample preparation protocol and relatively simple device, a field-use NASBA detection procedure is very feasible for specific applications, such as confirmation of a particular seafood product.